Testicular germ cell tumors (TGCTs) are the most common carcinomas of young men. Currently available cisplatin-based cytotoxic therapies result in substantial acute and life-long side effects that greatly impact quality of life. In addition, 15- 20% of cases are refractory or relapse despite cisplatin therapy and are fatal. There are currently no reliable prognostic makers to predict non-responders. This application is based on our novel observations that pluripotent embryonal carcinoma (EC), the stem cells of TGCTs, are exquisitely sensitive to low dose demethylation treatment with 5-aza deoxycytidine (5-aza) and that low dose 5-aza is effective in mediating promoter demethylation and p53 target gene expression. The 5-aza hypersensitivity extends to cisplatin resistant EC and 5-aza pretreatment can restore cisplatin sensitivity to refractory cells. Together, this work provides the rationale for our recently initiated and highly promising phase I clinical trial to study the novel 5-aza prodrug SGI-110 in cisplatin refractory TGCT patients. The objective of this proposal is to define the precise molecular mechanism(s) of TGCT hypersensitivity to DNA methylation therapy in order to provide the rationale and biomarkers of response to support future epigenetic-based therapy trials to treat testicular cancer and other solid tumors. The hypothesis is that TGCTs, even those refractory to standard platinum based chemotherapy, are hypersensitive to low dose treatment with DNA methylation inhibitors and can also be resensitized to cisplatin based-therapy. We also hypothesize that specific epigenetic features of pluripotent TGCT cells make them vulnerable to DNA methylation inhibitor therapy and that at least some of these features could apply to rare cancer initiating cells in somatic solid tumors. Our studies will use a combination of cell culture, human xenograft and PDX models, and primary patient samples including samples from the ongoing Phase I trial of SGI-110 in cisplatin refractory TGCTs. Aim 1 will determine whether 5-aza/SGI-100 and cisplatin sensitivity in TGCTs is due to specific alterations in genome-wide methylation and chromatin accessibility. Aim 2 will determine whether the mechanism of 5-aza/SGI-110 hypersensitivity in TGCT cells is due to hyperactivation of immune surveillance pathways. Aim 3 will validate these findings in PDX and orthotopic xenograft models and in primary TGCTs including tumors from an ongoing SGI-110 phase I therapeutic trial. This project teams molecular and epigenetic biologists with an esteemed clinically oriented research group that will set the stage for interventions targeting the TGCT epigenome and guide and impact the design of future clinical investigations.